Principle of Allele-Specific PCR Assay

In the allele-specific PCR assay for distinguishing between CRM197 and wild-type tox gene alleles，genomic DNA is PCR-amplified with four oligonucleotide primers：BTOX5P1（forward），JET1372（reverse），197M（forward），and 197N（reverse）.BTOX5P1 and JET1372 are the flanking primers，while the 197M and 197N primers are specific for the CRM197 mutant and the wild-type alleles，respectively，with each matching allele-specific nucleotide at its 3-end（see Figure 3）.Three PCR products were expected with the above set of four primers：a 740-bp control product with BTOX5P1 and JET1372；a 242-bp CRM197-specific product with 197M and JET1372 showing the presence of CRM197 template；and a 547-bp wild-type-specific product with BTOX5P1 and 197N showing the presence of wild-type template（see Figure 3）.

Table 1　Precision of the AluI RFLP assay

ⓐTemplate samples obtained by mixing wild-type and CRM-Ref.DNAs，both at 0.1 ng/μL concentration.The samples were subjected to AluI RFLP assay.The details of the assay are as previously described under Materials and Methods，AluI RFLP quantitative assay section.The percentage（71+34 bp）was calculated after scanning of the negative of polyacrylamide gel，in which the AluI digests of PCR products were separated.
ⓑCalculated as：